Cholesterol ester is the major lipid species accumulated largely as cytosolic droplets in human atherosclerotic plaques. The formation of intracellular cholesterol esters is catalyzed by the enzyme acyl coenzyme A: cholesterol acyltransferase (ACAT). The molecular mechanism(s) for ACAT regulation is not understood at present. The availability of cell mutants defective in cholesterol ester formation may serve as valuable tool to study ACAT regulation. We have recently isolated two such mutants from mutagenized parental Chinese hamster ovary (CHO) cells. Pilot studies indicate that these are mutants deficient in ACAT activity. We ask support to perform the following experiments (1) To characterize these two mutants (#27 and #90). We will perform Km studies, heat inactivation studies, and chemical modification studies to test if #27 or #90 contains a structural gene mutation in ACAT. Cell hybridization experiments will be performed to test if the mutant phenotype in #27 or #90 is recessive, dominant, or codominant. (2) To isolate more cell mutants from a large population of mutagenized parental cells (greater than or equal to 400 x 10-6 cells). To classify these mutants as lysosomal mutants or as post-lysosomal mutants; among the post-lysosomal mutants, to classify them as ACAT mutants or as mutants that possess normal ACAT but fail to accumulate cholesterol ester. (3) Methods will be developed for isolating spontaneous revertants from mutants defective in cholesterol ester formation. These methods will also be employed for isolating transformants from these mutants transfected with human DNA to correct the cholesterol ester deficiency. (4) The well-characterized ACAT deficient mutants will be used as a tool to study the role of ACAT in intracellular cholesterol homeostasis. We will perform experiments to seek an answer to the following question: What are the fates of LDL-bound cholesterol in a cell which lacks ACAT? (5) Using a well-characterized cell mutant possessing the structural gene defect for ACAT as the recipient, DNA mediated gene transfer experiments will be performed to transfect the mutant with human high molecular weight DNA. The successful secondary or tertiary transformants will be isolated and characterized biochemically with respect to sterol-dependent regulation of ACAT activity. In addition, the size limit of a functionally expressed Human ACAT genomic DNA will be estimated by standard methods.